Body weight, plasma and hepatic cholesterol, liver function and histology, gallbladder examination and histology

-Mice were obtained from the Jackson Laboratory and adapted in our animal room for at least one week on a commercial chow diet.

-At 9-10 weeks (whenever possible, see exact age in file), mice were weighed and placed on an high-fat diet (containing 15% butter fat, 1% cholesterol and 0.5% cholic acid) (Khanuja, 1995).

-After 8 weeks on the lithogenic diet, body weight was recorded and mice were fasted for four hours before blood collection in ice cold tubes containing EDTA through the retroorbital plexus. Blood was kept on ice until separation of plasma by centrifugation. Mice were sacrificed by cervical dislocation. Following a laparotomy, tissues were collected, weighted (liver) and processed as follows:

1. The heart and aorta were washed in saline, immersed in a 4% formalin solution overnight and transferred to a 10% formalin solution. Tissues were then processed as previously described (Paigen et al. 1987). In brief, hearts were embedded in 25% gelatin, and sectioned in a cryostat at -25 °C. Fatty streak aortic lesions size was average from 5 aortic sections of 10 microm cut at 80 microm intervals. Staining was performed with Oil Red (neutral lipids) and hematoxylin and counterstain with light green. The mean size of lesion per section as well as the Log of (mean size of lesion per section +1) are reported.

2. The gallbladder was excised after a ligation of the cystic duct and its volume assessed by gravimetry, assuming a density of tissue and bile of 1g/ml. Fresh gallbladder bile was immediately examined under polarizing light microscopy as previously described in details (Wang et al, 1997). Score were established by a single investigator on a scale of 0 to 4 for presence of mucin gel, crystallization through the "anhydrous" cholesterol pathway (presence of arc and filaments) and through the liquid crystal pathway (presence of small liquid crystal, aggregated liquid crystal and fused liquid crystals). Cholesterol monohydrates, common to both pathways and highly bi-refringent, were also scored. Sandy stones (early, soft stones, partially refringent) and true gallstones (completely non-refringent) were counted under the microscope. Mean number of stone per mice and prevalence of gallstones are calculated.

3. For three mice per sex per group, the gallbladder wall and a frontal hepatic lobe were processed for histology. In brief, tissues were immersed in Bouin's fixative, dehydrated in ethanol and stained with hematoxylin-eosin. Sections were scored from 0 to 4 by an expert histologist for inflammation (refers to infiltration of mixed inflammatory cells); microvacuoles (refers to presence of small vacuoles in the hepatocyte cytoplasm) and macrovacuoles (refers to large single vacuoles that replace the hepatocyte cytoplasm). Gallbladder histology is still to be finished.

4. Plasma was analyzed fresh for alanine aminotransferase, total cholesterol, HDL-cholesterol, total bile salts using Beckman CX5 Delta Chemistry Analyzer and reagents and procedure as supplied by the manufacturer except for bile acids (Sigma-Aldrich).

5. Liver was frozen in liquid nitrogen until processed for hepatic determination of cholesterol. Tissue was homogeneized, lipids extracted through a Folch procedure and hepatic cholesterol was determined using cholesterol oxidase (Sigma) while free cholesterol was determined by high pressure liquid chromatography (HPLC) as previously described (Lammert et al. 1999; Wang et al. 1999). Cholesterol esters were calculated by substraction of free cholesterol from total cholesterol.